AbstractThis study aimed to investigate the antioxidant and immune responses of crayfish Procambarus clarkii to V. parahaemolyticus infection through a comparative analysis of the immune index, antioxidant capacity, histomorphology, and transcriptome data of V. parahaemolyticus-infected and PBS-treated P. clarkii. The findings indicated that V. parahaemolyticus infection resulted in intestinal cell necrosis, a reduction in the number and height of intestinal villi, and an increased absorption rate of the intestinal jejunum. The prolonged infection time of V. parahaemolyticus resulted in an initial increase in intestinal immunity and antioxidant enzyme activity, followed by a subsequent decrease, ultimately returning to levels similar to those in the control group. The intestinal transcriptional response to V. parahaemolyticus infection was assessed using RNA sequencing (RNA-seq). A total of 27,867 genes were significantly assembled, and 2124 genes showed differential expression, including 849 upregulated and 1275 downregulated genes. These genes were primarily enriched in KEGG pathways such as Ribosome, Oxidative phosphorylation, Phagosome, Inositol phosphate metabolism, and adrenergic signaling in cardiomyocytes. Simultaneously, ATP synthesis-coupled electron and proton transport, mitochondrial electron transport, and other energy metabolism pathways were significantly enriched in the Gene Ontology (GO) database. qRT-PCR analysis confirmed the reliability of the RNA-seq results for five randomly selected differentially expressed genes (DEGs). Our experimental data contributes to the transcriptome database of P. clarkii, enhances our understanding of the immune and antioxidant defense mechanisms of P. clarkii, and provides a foundation for future research on disease control in crayfish aquaculture.